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FITC1 Sigma

FluoroTag FITC Conjugation Kit

Synonym: FITC

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Description

Frequently Asked Questions

Frequently Asked Questions are available for this Product.

Analysis Note

Procedure
1. Dissolve protein and FITC in carbonate-bicarbonate buffer.
2. Slowly add FITC to protein with stirring. Cover with foil and stir 2 hours at room temperature.
3. Separate conjugate from free FITC on G-25 column. Collect fractions.
4. Pool fractions containing conjugate.
5. Determine F/P ratio of conjugate spectrophotometrically.
6. Stabilize with 1% bovine serum albumin and 0.1% sodium azide and store at 0-5 °C.

Features and Benefits

• Suitable for both small (1 mg) and large (5 mg) scale conjugations
• Completely aqueous procedure - no DMF needed
• Fast gel filtration separation of conjugate from excess FITC
• Complete protocols for conjugation and F/P ratio determination
• Sufficient reagents for at least 5 conjugations of 5 mg protein each and for optimization of F/P ratio before scale-up
• References for applications and protocols

Principle

Sigma offers a convenient kit for preparing FITC-labeled antibodies. Fluorescein isothiocyanate (FITC), Isomer 1, is a widely used fluorophore, popular because of its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through free amino groups of proteins or peptides, forming a stable thiourea bond (see reaction). FITC conjugates of antibodies, lectins, hormones, and growth factors have been used in a variety of immunohistochemical and flow cytometry applications. The protocols have been optimized for antibodies, but may be adapted to other proteins by the end user.

Application

The Flourotag FITC Conjugation Kit can be used for Immunohistochemisty and Immunocytochemistry using Flow Cytometry. It is also be used for the conjugation of FITC to peptide hormones, cytokines, growth factors and proteins.

Legal Information

FluoroTag is a trademark of Sigma-Aldrich Co. LLC

Price and Availability


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Kit component only

Description

Product #

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0.1 M Carbonate-bicarbonate buffer capsule, pH 9.0 5 capsules SDS    

Standard component

Description

Product #

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Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder 5 x 2 mg SDS F7250
Phosphate buffered saline, powder, pH 7.4, for preparing 1 L solutions 5 pkg SDS P3813
Sephadex G-25 column, 3.5 mL 1 SDS    
Sephadex G-25 column, 9.1 mL 1 SDS    
Safety & Documentation

Safety Information

Symbol 
GHS07  GHS07
Signal word 
Warning
Hazard statements 
Precautionary statements 
RIDADR 
NONH for all modes of transport

Frequently Asked Questions

Which document(s) contains shelf-life or expiration date information for a given product?
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis. These documents are located on the product detail page under Useful Links & Tools. Click on the following link to search for a Certificate of Analysis. Please click the following link to see the details on our Product Dating Information.
How do I get lot-specific information or a Certificate of Analysis?
A Certificate of Analysis is available by lot number and can be obtained through our Advanced Search Option: http://www.sigmaaldrich.com/catalog/AdvancedSearchPage.do
Can Product FITC1, FluoroTag™ FITC Conjugation Kit, be used with antibodies at concentrations less than 5 mg/mL?
You can scale down the reaction for labeling.  A possible problem occurs in using the column to remove the free FITC.  If the volume is too small, the column cannot be used.  We would recommend using dialysis to remove the free (unconjugated) FITC.
Can the columns in Product FITC1, FluoroTag™ FITC Conjugation Kit, be reused?
Wash the column with 35 ml (10 X column volumes) of PBS to remove unbound fluorophore. This is sufficient to regenerate the column.  For prolonged storage, wash the column with 10 mL of PBS containing 0.05% sodium azide and store capped at 2-8 °C, with about 1 mL buffer above the gel.
When using Product FITC1, FluoroTag™ FITC Conjugation Kit, should I adjust the dye:protein ratio for the size of the protein being labeled?
Adjustments for dye/protein ratios should only be made for very small proteins, i.e. < 25 kDa. Here, you should use less protein per dye portion. In many cases, this weight amount of protein is IgG with MW 150 kDa, and the kit would provide a molar excess of dye to provide attachment of a few dyes per IgG. If one wants to attempt labeling of a very small protein using the same weight ratio, there is a good chance there would not be enough dye/protein.
My antibody is in solution containing azide.  Can I use the antibody directly in the reaction when using Product FITC1, FluoroTag™ FITC Conjugation Kit?
It is not recommended to have azide in the reaction mix. If the buffer contains amines or sodium azide, dialyze protein solution (1 mL) against PBS, pH 7.4 (approximately 1000 mL), overnight at 2-8 °C.
How do I find price and availability?
There are several ways to find pricing and availability for our products.  Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote.  USA customers:  1-800-325-3010 or view local office numbers. 
What is the Department of Transportation shipping information for this product?
Transportation information can be found in Section 14 of the product's (M)SDS. To access the shipping information for this material, use the link on the product detail page for the product, or search here. 
My question is not addressed here, how can I contact Technical Service for assistance?
Use the option to the right to "Ask a Question" by email of a Technical Service Scientist.
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Protocols & Articles

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References

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